ABSTRACT
Cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the oxyfunctionalization of non-activated carbon-hydrogen bonds. Most CYPs rely on electron transport proteins as redox partners. In this study, the ferredoxin reductase (FdR) and ferredoxin (FD) for a cytochrome P450 monooxygenase from Acinetobacter sp. OC4 are investigated. Upon heterologous production of both proteins independently in Escherichia coli, spectral analysis showed their reduction capability towards reporter electron acceptors, e. g., cytochrome c. The individual proteins' specific activity towards cytochrome c reduction was 25â U mg-1. Furthermore, the possibility to enhance electron transfer by artificial fusion of the units was elucidated. FdR and FD were linked by helical linkers [EAAAK]n, flexible glycine linkers [GGGGS]n or rigid proline linkers [EPPPP]n of n=1-4 sequence repetitions. The system with a glycine linker (n=4) reached an appreciable specific activity of 19â U mg-1 towards cytochrome c. Moreover, their ability to drive different members of the CYP153A subfamily is demonstrated. By creating artificial self-sufficient P450s with FdR, FD, and a panel of four CYP153A representatives, effective hydroxylation of n-hexane in a whole-cell system was achieved. The results indicate this protein combination to constitute a functional and versatile surrogate electron transport system for this subfamily.
ABSTRACT
Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows NAD(P)+ recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreductase (AzoRo) of Rhodococcus opacus 1CP and FDH (FDHC23S and FDHC23SD195QY196H ) of Candida boidinii in different positions with His-tag as the linker. The position affected enzyme activities as AzoRo activity decreased by 20-fold when it is in the N-terminus of the fusion protein. FDHC23S +AzoRo was the most active construct and was further characterized. Enzymatic activities of FDHC23S +AzoRo decreased compared to parental enzymes but showed improved substrate scope - accepting bulkier dyes. Moreover, pH has an influence on the stability and activity of the fusion protein because at pHâ 6 (pH that is suboptimal for FDH), the dye reduction decreased to more than 50 % and this could be attributed to the impaired NADH supply for the AzoRo part.
Subject(s)
Formate Dehydrogenases , NAD , Biocatalysis , Coloring Agents , Formate Dehydrogenases/chemistry , NAD/metabolism , Nitroreductases/metabolismABSTRACT
[This corrects the article DOI: 10.1007/s13205-020-2136-3.].
ABSTRACT
In the present study, we report the draft genome of soil isolate DP-K7 that has the potential to degrade methyl red. The 16S rRNA gene sequencing and whole-genome analysis exposed that the bacterial strain DP-K7 belongs to the species Kocuria indica. The genome annotation of the strain DP-K7 through the bioinformatics tool "Prokka" showed that the genome contains 3,010,594 bp with 69.01% GC content. The genome comprises 57 contigs including 2 rRNA genes, 47 tRNA genes, and 2754 CDS. The plate and broth assay showed that the strain DP-K7 has the potential to utilize methyl red as the sole carbon source for growth. Indeed, the RP-HPLC analysis proved that the strain DP-K7 is capable of degrading methyl red. The genome BLAST against a characterized azoreductase (AzoB-Xenophilus azovorans KF46F) revealed the presence of two azoreductase-like genes (azoKi-1 and azoKi-2). The phylogenetic analysis of the primary amino acid sequence of characterized azoreductases suggested that AzoKi-1 and AzoKi-2 belong to members of the clade IV azoreductase, which are flavin-independent. The multiple sequence alignment of AzoKi-1 and AzoKi-2 with flavin-independent azoreductases showed the presence of NAD(P)H binding like motif (GxxGxxG). In addition, other genes coding for dye degrading enzymes (SodC, SodA, KatA, KatE, and DyP2) were also found in the genome supporting that the strain K. indica DP-K7 is a potential azo dye degrader.
